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p. 1
imageaide getting started although all possible care has been taken in the preparation of this publication spectronics corporation accepts no liability for any inaccuracies that may be found spectronics corporation reserves the right to make changes without notice both to this publication and to the product that it describes no part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording or otherwise without the prior permission of the copyright owner © synoptics limited 20002004[close]
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spectronics corporation 959 brush hollow road p box .o westbury new york 11590 tel 1-800-274-8888 fax 1-800-491-6868 website www.spectroline.com[close]
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table of contents introduction 11 analysis techniques 11 starting up imageaide 12 note on file formats 12 getting started with gel analysis 21 setting up imageaide for getting started 21 opening a secure sample file 22 saving a secure sample file 26 using gel windows 27 image pane 27 track label pane 28 description pane 28 profile pane 28 peak value pane 29 graphics pane 29 results pane 29 determining molecular weights 210 working with results 213 printing gel results 214 measuring quantities 215 matching peaks on different tracks 216 next steps 217 getting started with colony counting opening a secure sample file saving a secure sample file using colony counting windows setting the area in which colonies defining an exclude region are counted 31 31 34 35 36 37 imageaide user manual contents-1[close]
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contents changing the shape of an exclude region 39 deleting exclude regions 311 setting the parameters used to detect colonies 312 viewing colony counting results 313 defining the colony class boundary 314 adding and removing colonies manually 315 printing colony counting results 317 next steps 318 getting started with manual band quantification 41 opening a secure sample file 41 placing boxes on the sample 45 placing a rectangular box on the sample 45 placing a free hand box on the sample 47 adjusting the position of a box 49 adjusting the size and shape of boxes 49 removing a box from a manual band quantification sample 411 showing or hiding identification labels on the boxes 411 saving a secure sample file 412 assigning quantities to boxes 412 applying background correction to results 414 viewing manual band quantification results 415 defining an incidence condition 416 viewing the incidence matrix 417 printing manual band quantification results 418 next steps 419 getting started with spot blot analysis 51 opening a secure sample file 51 saving a secure sample file 55 assigning quantities to spot frames 56 applying background correction to results 57 viewing spot blot results 58 defining an incidence condition 510 viewing the incidence matrix 512 printing spot blot results 513 next steps 514 contents-2 imageaide user manual[close]
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introduction this manual shows you how to get started with imageaide however you will probably find that the few simple procedures it describes will cover nearly all you need to know to use imageaide in most circumstances for example imageaide provides many configuration options allowing you to control the way it works but you are unlikely to need to change these settings very often if at all since the default settings will cope with most situations if you do change the settings you can save them as the defaults to be used for new images imageaide also provides a range of tools to give you fine control in carrying out procedures manually or for editing the results of its automatic operations however you will probably only need to use them very occasionally since imageaide s automatic tools are so powerful and sensitive that they will handle all but the most exceptional situations where appropriate the manual will draw your attention to some of the features not covered in detail here with references to sections in the on-line help where you can find full instructions for how to carry out all imageaide operations analysis techniques imageaide supports gel analysis colony counting manual band quantification and spot blot analysis each of these is covered separately in the following chapters getting started with gel analysis chapter 2 getting started with colony counting chapter 3 getting started with manual band quantification chapter 4 getting started with spot blot analysis chapter 5 imageaide user manual 1-1[close]
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introductions pugnirat starting up imageaide to start up imageaide 1 click on the start button in the windows task bar and choose imageaide from the programs®spectronics submenu depending on options set in the program the user name dialog box may be displayed 2 if you have entered a imageaide user name previously you will be able to select it from the drop-down list box otherwise enter your name the user name is printed in reports and is recorded in document histories to keep track of who has created and changed files in imageaide you can uncheck show at program start if you don t want the user name dialog box displayed when the program starts the user who was logged in when the program was last closed will be logged in again automatically note on file formats imageaide stores data in a secure file format a .sgd file these files are also created by compatible acquisition programs such as imagesource the secure file format ensures that your data cannot be tampered with and that your results are both traceable and reproducible however you can also load other image files created by non-secure sources such as tiff or bmp images generated by a scanner into imageaide for analysis when you do this imageaide copies the original image and saves it in a secure file any changes you then make to the original image will have no effect on the image in the secure file 1-2 imageaide user manual[close]
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getting started with gel analysis this chapter shows you how to get started using imageaide for gel analysis see the on-line help for full details the chapter begins with a section explaining how to set up the program so that you can work through the rest of the chapter setting up imageaide for getting started this chapter assumes that imageaide is configured to carry out operations automatically if you have just installed imageaide this will be the case as the automatic options are on by default however if you are not using a new installation some of the automatic options may have been turned off so you should make sure they are all turned on before going any further to set up imageaide for automatic operation 1 choose configuration from the extras menu to display the general page in the imageaide configuration dialog box imageaide user manual 2-1[close]
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getting started with gel analysiso flmrucsaginep 2 make sure that auto match peaks auto locate tracks and auto locate peaks are selected 3 uncheck auto import methods 4 press ok to confirm your changes and close the dialog box opening a secure sample file note if you have started imageaide directly from imagesource the sample properties dialog box will be opened immediately and you can go straight to step 4 in the instructions below to open an existing secure sample file in imageaide 1 press the open button in the standard toolbar or choose open from the file menu note pressing the open button is a quick alternative to choosing the command from the menu throughout the rest of this manual if a button is shown beside an instruction you can press the button as an alternative to using the command you can carry out most of the common operations in imageaide by pressing buttons 2-2 imageaide user manual[close]
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y lhwdrasgniteg opening a secure sample file a standard windows open dialog box will be displayed note if you want to analyze images generated by non-secure sources for example a .tif or .bmp image file choose new import from the file menu this will create a new secure sample file and then display a standard windows open dialog box allowing you to select the required image file see the on-line help for details 2 use the dialog box to select the required secure gel sample file 3 press open note if you are opening an analyzed sample file one that has been previously saved in imageaide the sample will be opened immediately in a gel window if you have set auto import methods and you have selected a sample file as the source for the settings to import the import method dialog box will open so that you can choose which settings to import see the on-line help for details imageaide user manual 2-3[close]
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getting started with gel analysiso flmrucsaginep the sample properties dialog box will open with the image loaded 4 check that the analysis type is set to gel this is the default analysis type when you first install imageaide but you can choose a different default analysis type using configuration in the extras menu 5 press one of the electrophoresis direction radio buttons to specify the electrophoresis direction in the image the default is down note the red box on the image in the preview pane defines the area of interest imageaide will only locate tracks within this area by default the area of interest includes most of the image so you should not need to adjust it however you can move or reshape the area of interest if required or define more than one area of interest see the on-line help for details 2-4 imageaide user manual[close]
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y lhwdrasgniteg opening a secure sample file you shouldn t need to change any of the other settings in particular imageaide automatically detects the image type unless you are opening a multi-layer sample you should leave the numbers of columns and rows set to 1 see the on-line help for details if you want to define more than one area of interest you should leave locate tracks automatically selected and hint unchecked this means imageaide will use fully automatic track location which is the recommended option as in nearly all cases it gives very accurate results in the shortest time 6 press ok there will be a short pause while imageaide first automatically locates the tracks on the gel image and then locates the peaks along each of the tracks the results will be displayed in a gel window for more detailed instructions and related topics see the following topics in the reference ® gel analysis ® how to ® create and work with secure sample files section of the on-line help opening a secure sample file creating a new secure sample file using the browser acquiring an image to create a secure sample file setting the integration parameters imageaide user manual 2-5[close]
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getting started with gel analysiss flpmrucesgniva saving a secure sample file once you have created the new secure sample file you should save it if you will need to use it again saving the file saves all the track and peak positions and analysis results etc with the image to save a secure sample with a new name 1 choose save as from the file menu to display a standard windows save as dialog box 2 use the dialog box to select a folder in which to save the sample 3 enter a file name for the sample 4 press save to save the sample in the file to save a previously saved secure sample file using the same name choose save from the file menu if the secure sample file has not been saved previously the save as dialog box will be displayed so that you can enter a name see above 2-6 imageaide user manual[close]
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y lhwdrasgniteg using gel windows using gel windows once you have opened a secure gel sample file it will be displayed in a gel window image pane track label pane description pane profile pane peak value pane reset brightness image brightness adjustment reset contrast image contrast adjustment reset gamma gamma correction adjustment graphics pane results pane the following subsections give a quick overview of the different panes in the gel window see the on-line help for full details image pane the image pane shows the image with the tracks and peak positions and bounds overlaid on it note that the peak and bounds markers are curved grimaced to match the data imageaide user manual 2-7[close]
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getting started with gel analysisu odwleggnis when you match the peaks between tracks lines will be shown joining the matching peaks you can choose whether to show each of these features using commands in the view menu and the color to use for them using configuration in the extras menu you can select a peak in the image pane by clicking on it this also selects the track you can right-click on a peak in the image pane to display a menu for working with the selected peak most of the commands also appear on the peak menu track label pane the track label pane shows the number of each track and whether it has been disabled the number is shown in gray selected as a molecular weight standard mws selected as a matching standard ms or selected as the active matching standard ms the picture shows a newly analyzed image so none of these apply you can select a track in the track label pane by clicking on it you can right-click on a track in the track label pane to display a menu for working with the selected track most of the commands also appear on the track menu description pane the description pane shows a description of the selected track use description in the tracks menu to enter or edit the description profile pane the profile pane shows the profile for the selected track the position and boundaries of each peak are shown on the profile if you choose to make one of the tracks a matching standard its profile will be overlaid on the profile for other tracks 2-8 imageaide user manual[close]
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y lhwdrasgniteg using gel windows you can choose the colors used for any of these features using configuration in the extras menu you can right-click on a peak in the profile pane to display a menu for working with the selected peak most of the commands also appear on the peak menu peak value pane the peak value pane shows data for the peaks shown in the profile pane the data shown depends on what is selected in the graphics pane shown in the bottom left-hand corner of the gel window molecular weights are shown if the molecular weight calibration curve is selected quantities are shown if the quantity calibration curve is selected peak positions are shown if the dendrogram is selected graphics pane the graphics pane can show the molecular weight calibration curve for the selected track the quantity calibration curve for the selected track the dendrogram showing relationships between matched tracks the example picture shows a newly analyzed image so all of these are blank results pane the results pane can show tables with results for selected track results for all tracks matching comparisons showing results for matched peaks matching matrix showing which peaks matched matching coefficients showing the matching between tracks imageaide user manual 2-9[close]
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getting started with gel analysisd shwauclognimrte right-clicking on the results pane displays a menu that allows you to choose what fields are displayed in each of the first three tables listed above determining molecular weights in order to determine the molecular weights of peaks on the tracks you must assign molecular weights to known peaks on one or more standard tracks imageaide is supplied with a library of commonly used molecular weight standards you can use these as they are edit them or add your own standards if you have a track on the gel corresponding to one of these standards you can assign the molecular weights from the standard in the library to the peaks in the track alternatively you can enter individual molecular weights and assign them to individual peaks manually the following instructions show you how to assign the molecular weights from a standard in the library see the cross-references listed at the end of this section for more information about creating and editing molecular weight libraries and assigning molecular weights to assign molecular weights to the peaks on a standard track 1 click in the standard track to select it 2 click on the peak in the track corresponding to the first molecular weight in the standard 2-10 imageaide user manual[close]
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y lhwdrasgniteg determining molecular weights 3 choose mw standard from the track menu or the pop-up menu displayed by right-clicking in the track label to define the track as a molecular weight standard and display the assign molecular weight dialog box it will open at the from standard page arrow buttons to select the track peak standards drop-down list details of selected standard 4 select the required molecular weight standard from the standards drop-down list 5 check that the first standard shown in the standard details list corresponds to the selected peak if it doesn t use the arrow buttons to select the peak that does correspond to the first standard 6 press assign from standard 7 press ok imageaide user manual 2-11[close]
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getting started with gel analysisd shwauclognimrte provided molecular weight is selected in the view menu the assigned molecular weights will appear in the peak value pane next to the relevant peaks in the profile pane the peak markers for peaks on the standard track that have molecular weights assigned to them will have a short marker drawn across them 2-12 imageaide user manual[close]
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y lhwdrasgniteg working with results the mw calibration page in the gel window s graphics pane will show the calibration points and the selected calibration curve joining them the vertical axis shows molecular weight the scale may be linear or logarithmic depending on the choice made using configuration in the extras menu the horizontal scale shows pixel position down the track the assigned molecular weights will also appear in the tables in the results pane assigned molecular weights are shown in red calculated molecular weights are shown in black if you select another track the molecular weights calculated for the peaks in that track will be shown in the peak value pane and in the results pane for more detailed instructions and related topics see the reference ® gel analysis ® how to ® create molecular weight calibrations section in the on-line help working with results once imageaide has analyzed the gel image you can view the results tables in the results pane click in the pane and choose maximize pane from the view menu to expand the pane to fill the window or right-click in the pane and choose maximize pane from the pop-up menu choose the command again to return to the normal view you can choose what information to show in the tables by right-clicking in the results pane and selecting the columns you want to display imageaide user manual 2-13[close]
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getting started with gel analysisw lusehtwgnikro printing gel results to print a report showing gel results 1 choose report setup from the file menu to display the report setup dialog box note if the paper selected for the currently selected printer is less than 150 mm in either dimension for example if you are using a video printer a simplified report containing the image and some brief identification and image information will be printed the settings in the report setup dialog box have no effect on the content of this simplified report 2 check the boxes for the items you want to include in the report 3 press ok to close the dialog box 4 choose print from the file menu to display the print dialog box so that you can choose print options this is a standard windows dialog box 5 choose the print options you require and press ok to print the report 2-14 imageaide user manual[close]
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y lhwdrasgniteg measuring quantities note see the reference ® gel analysis ® how to ® printing reports gel results topic in the on-line help for further details you can also export results from the tables in the results pane to excel save results in a comma separated values format file and copy a picture of the image to the clipboard see the on-line help for details measuring quantities to calibrate the quantity measurements for the peaks you must know the quantity corresponding to at least one peak and then assign this quantity to the peak in imageaide to assign a calibration quantity to a peak 1 click on the required peak in the image pane to select it 2 choose assign quantity from the peak menu or the pop-up menu displayed when you right-click on the peak to display the assign calibrated quantities dialog box the controls at the bottom of the dialog box are disabled if you choose to use the same calibration for all tracks this is the default you can use quantity calibration in the edit menu to allow separate calibrations for each track 3 enter the known quantity for the peak in the calibrated quantity box 4 press ok imageaide user manual 2-15[close]
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getting started with gel analysism rfdoskepgnihcta the peak marker in the image pane will have a short line across it to show that it is a quantity calibration standard the graphics pane will show the quantity calibration curve derived from this calibration point and the peak value pane will show the quantities calculated from the calibration curve the results pane will also show the calibrated quantity if you choose to display the quantity column in the tables right-click in the results pane and choose calibration quantity for more detailed instructions see the reference ® gel analysis ® how to ® create quantity calibrations section in the on-line help matching peaks on different tracks note this section only applies if you have purchased the imageaide match software option imageaide allows you to find peaks on other tracks that match the peaks on a standard track to define a track as the active matching standard 1 click on the track in the image pane to select it 2 choose matching standard from the track menu the track label will show ms the ms means it is a matching standard the means it is the active matching standard any previous active matching standard will now show ms to show that it is still a matching standard but no longer the active matching standard provided auto match peaks is set in the imageaide configuration dialog box you checked this at the beginning of the getting started sequence and peak matching is selected in the view menu match lines will be drawn on the image between the peaks on the matching standard and any matching peaks on the other tracks for more detailed instructions and related topics see the reference ® gel analysis ® how to ® match peaks on different tracks section in the on-line help 2-16 imageaide user manual[close]
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y lhwdrasgniteg next steps next steps this completes the getting started with gel analysis chapter if you follow the instructions given in this chapter you should now be able to get started working productively with imageaide to analyze gels as you will have noticed from the number of cross-references to the on-line help for further details imageaide offers many other features and refinements but as long as you are generally aware of what is available you can probably leave the details until you need to use them imageaide user manual 2-17[close]
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getting started with colony counting this chapter shows you how to get started using imageaide for colony counting see the on-line help for full details opening a secure sample file note if you have started imageaide directly from imagesource the sample properties dialog box will be opened immediately and you can go straight to step 4 in the instructions below to open an existing secure sample file in imageaide 1 press the open button in the standard toolbar or choose open from the file menu note pressing the open button is a quick alternative to choosing the command from the menu throughout the rest of this manual if a button is shown beside an instruction you can press the button as an alternative to using the command in the instruction you can carry out most of the common operations in imageaide by pressing buttons imageaide user manual 3-1[close]
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